Methods and compositions for treating multiple sclerosis and related disorders

ABSTRACT

This disclosure provides therapeutic compositions and methods for treating multiple sclerosis or a multiple sclerosis-related disorder in a subject in need thereof comprising administering an effective amount of an antigen-MHC-nanoparticle complex to the subject, wherein the antigen is a multiple sclerosis-related antigen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/433,898, filed Feb. 15, 2017, which is a continuation of U.S.application Ser. No. 14/684,153, filed Apr. 10, 2015, which is acontinuation of International Application No. PCT/IB2013/003033, filedOct. 11, 2013, which in turn is a continuation-in-part of U.S.application Ser. No. 13/830,521, filed Mar. 14, 2013, and claimspriority under 35 U.S.C. § 119 (e) to U.S. Provisional Application No.61/712,733, filed Oct. 11, 2012, the content of each of which isincorporated by reference in its entirety into the present disclosure.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 22, 2014, isnamed 378701-0610_SL.txt and is 11,284 bytes in size.

FIELD OF DISCLOSURE

This disclosure is directed to compositions and methods related toimmunotherapy and medicine. In particular, this disclosure is related totherapeutics for the treatment of multiple sclerosis and relateddisorders.

BACKGROUND

Multiple sclerosis (MS) is a potentially debilitating disease in whichthe body's immune system eats away at the protective sheath that coversyour nerves. This interferes with the communication between the brainand the rest of the body. Ultimately, this may result in deteriorationof the nerves themselves, a process that is not reversible.

Statistics show that approximately 250,000 to 350,000 people in theUnited States have been diagnosed with this disease. Symptoms varywidely, depending on the amount of damage and which nerves are affected.People with severe cases of multiple sclerosis may lose the ability towalk or speak. Symptoms of MS include numbness in the arms or legs,pain, loss of vision, muscle weakness or tremors, paralysis, vertigo,fatigue, difficulty with speech, bladder dysfunction, depression,hearing loss, and itching.

There is no cure for MS, but certain medications have been found to easeMS attacks and possibly slow the disease. Treatments attempt to returnfunction after an attack, prevent new attacks, and prevent disability.MS medications can have adverse effects or be poorly tolerated.Accordingly, there is a need in the art for better-tolorated, moreeffective therapies for MS.

SUMMARY

In response to a need in the art, described herein are therapeuticmethods and compositions for treating or preventing multiple sclerosisor multiple sclerosis-related disorders. One aspect of the disclosurerelates to a method for expanding and/or developing populations ofanti-pathogenic autoreactive T-cells in a subject with multiplesclerosis or a multiple sclerosis-related disorder which methodcomprises, consists essentially of or yet further consists ofadministering to that subject an antigen-MHC-nanoparticle complex,wherein the antigen is a multiple sclerosis-related antigen.

A further aspect relates to a method for treating multiple sclerosis ora multiple sclerosis related disorder in a subject in need thereofcomprising, consisting essentially of or yet further consisting ofadministering an effective amount of an antigen-MHC-nanoparticle complexto the subject, wherein the antigen is a multiple sclerosis-relatedantigen. Also provided is the use of an antigen-MHC-nanoparticle complexin the preparation of a medicament for the treatment of multiplesclerosis or for expanding and/or developing populations ofanti-pathogenic autoreactive T-cells, wherein the antigen is a multiplesclerosis-related antigen.

Other aspects relate to a complex comprising, consisting essentially ofor yet further consisting of, a nanoparticle; a MHC protein; and amultiple sclerosis-related antigen. Also provided are compositionscomprising, consisting essentially of, or yet further consisting of, theantigen-MHC-nanoparticle as described herein and a carrier. Yet furtheraspects relate to kits comprising, consisting essentially of, or yetfurther consisting of the antigen-MHC-nanoparticle compositionsdescribed herein and instructions for use.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows that pMHC class II-NP therapy reduces the severity ofestablished EAE in C57BL/6 mice. B6 mice were immunized with pMOG₃₅₋₅₅in CFA and treated with pertussis toxin i.v. Mice were scored for signsof EAE using established criteria over a 15-point scale. Affected micewere treated with two weekly doses of 7.5-22.5 ug ofpMOG₃₈₋₄₉/IA^(b)-coated NPs, beginning 21 days after immunization.

FIG. 2 demonstrates that mice with chronic EAE treated with pMHC classII-NP therapy (pMOG₃₈₋₄₉/1-A^(b)-coated NPs) have increased weightcompared to the untreated EAE mice.

FIG. 3 is a photagraph of treated and untreated mice with EAE. Treatedmice (NAVACIM) appear healthier than untreated mice.

FIG. 4 shows the systemic expansion of cognate autoregulatory CD4+T-cells by pMOG₃₈₋₄₉/IA^(b)-coated NPs in EAE-affected C57BL/6 mice. Themagnitude of expansion in this model is comparable to that seen in NODmice treated with type 1 diabetes-relevant pMHC class II-coated NPs(See, for example, U.S. Pat. No. 8,354,110, which is herein incorporatedby reference in its entirety.

FIG. 5 depicts the spinal cords of untreated mice. The untreated micedisplayed significant demyelination and dense mononuclear cellinfiltrates of the white matter.

FIG. 6 depicts the spinal cords of mice treated withpMOG₃₈₋₄₉/IA^(b)-NPs. pMHC-NP-treated mice had significantly lessdemyelination and mononuclear cell infiltrates.

FIG. 7 shows representative examples for the spinal cord edges of 2untreated EAE mice.

FIG. 8 shows representative examples for the spinal cord edges of 2 EAEmice treated with pMOG₃₈₋₄₉/IA^(b)-NPs. pMHC-NP-treated mice havesignificantly less demyelination as well as lower mononuclear cellinfiltration.

FIGS. 9A-9B show the protein (SEQ ID NO: 2) and DNA (SEQ ID NO: 3)sequences of pMOG₃₆₋₅₅-I-Abeta (b)-C-Jun construct (SEQ ID NOS: 2-3,respectively, in order of appearance). The sequences of individualcomponents in the fusion protein are HA leader (underlined) followed bypMOG₃₈₋₄₉_peptide sequence (double underlined), I-Abeta (b) (dottedunderlined) and C-Jun (shaded) sequences. GS linkers are nothighlighted. FIG. 9A depicts base pairs 849 through 1500 of SEQ ID NO: 3and the first 216 amino acids of SEQ ID NO:2. FIG. 9B depicts base pairs1501 through 1734 of SEQ ID NO: 3 and the final 74 amino acids of SEQ IDNO:2.

FIG. 10 is a DNA map of the antigen-containing vector. DNA constructsites encoding HA leader-I-Aalpha (b)-C-Fos-BirA-His×6 fusion protein(284 a.a) (“His×6” disclosed as SEQ ID NO: 18) was cloned into pMT/V5fly cell expression vector between Nco 1 (854) to Xba I (1711). Thefusion protein includes I-Aalpha (d) (195 a.a.), followed by C-Fosthough a GS linker (6 a.a.), and then BirA sequence and 6×His (SEQ IDNO: 18).

FIG. 11 shows representative TEM image of pMHC-coated gold NPs (˜14 nm)concentrated at high densities (˜5×10¹³/ml) and monodispersed. Mag:50,000×.

FIG. 12 shows the effects of pMHC (GNP) dose and pMHC valency on theagonistic properties of pMHC-coated NPs. The Figure compares the amountsof IFNγ secreted by cognate 8.3-CD8+ T-cells in response to twodifferent pMHC-NP samples (both consisting of ˜2×10¹³ NPs of 14 nm indiameter/ml). Au-022410 and Au-21910 carried ˜250 and ˜120 pMHCs/NP,respectively. Au-011810-C carried ˜120 control pMHCs/NP.

FIG. 13 demonstrates the pMHC-NP-induced secretion of IFNγ by 8.3-CD8+ Tcells as a function of pMHC valency. 8.3-CD8+ T-cells (2.5×10⁵ cells/ml)were cultured with increasing numbers of NPs coated with three differentIGRP₂₀₆₋₂₁₄/K^(d) valencies. IGRP₂₀₆₋₂₁₄ comprises the antigenic peptideV YLKTN VFL (SEQ ID NO: 19).

FIG. 14 shows that the lower agonistic activity of pMHC-NPs can becompensated by increasing the pMHC-NP density but only above a thresholdof pMHC valency. Graph compares the agonistic activity of threedifferent pMHC-NP preparations (carrying three different valencies ofpMHC) over a range of NP densities. Note that NPs carrying 8 pMHCs,unlike those carrying 11 pMHCs, cannot adequately trigger IFNγ secretioneven at high pMHC-NP densities, as compared to NPs carrying 11 and 54pMHCs per NP.

FIG. 15 shows the effects of pMHC valency threshold on the agonisticproperties of pMHC-NPs as a function of total pMHC input.

FIG. 16 shows the effects of pMHC valency on the agonistic activity ofpMHC-NPs produced with larger iron oxide NP cores.

FIG. 17 shows the effect of size on agonistic activity. Au-0224-15 were14 nm GNPs coated with a relatively low pMHC valency but prepared at ahigh density; Au-0323-40 were 40 nm GNPs coated with high pMHC valencybut at low density. Au-0224-15 had superior agonistic activity than theAu-0323-40 sample.

FIG. 18 shows the effect of protective PEGs on the function ofpMHC-GNPs. Au-021910 consisted of ˜2×10¹³ GNPs of 14 nm in diameter/mlprotected by 2 kD thiol-PEGs and coated with ˜120 pMHCs/GNP. Au-012810GNPs (also ˜2×10¹³ 14 nm GNPs/ml) were protected by 5 kD thiol-PEGs andwere coated with ˜175 pMHCs/GNP. Sample Au-021910 had superior agonisticactivity.

FIG. 19 shows the Efficient expansion of NRP-V7-reactive CD8+ T-cells byNRP-V7/Kd-coated gold NPs. 3×10¹² NPs (˜10 nm in size) carrying 25 μg ofpMHC (150 pMHC/NP) were used. Pre-diabetic 10 wk-old NOD mice weretreated with two weekly injections of NRP-V7/kd-coated gold NPs for 5weeks. TUM/Kd tetramer is a negative control. Each column of panelscorresponds to a different mouse.

FIG. 20 depicts the large expansion of cognate CD8+ T-cells in micetreated with pMHC-coated NPs. 3×10¹² IGRP₂₀₆₋₂₁₄/K^(d)-NPs (˜10 nm insize) carrying 25 μg of pMHC (150 pMHC/NP) were used. Upper panel:profile of a mouse sacrificed after 4 doses. Bottom panel: profile oftwo different mice after 10 injections (blood only).

FIG. 21 demonstrates that the same principles for pMHC class I-Npcomplexes apply to pMHC class II-coated Nps (sec FIG. 18). Note thatpMIIC class II pMHC-Nps (BDC2.5mi-coated 6-8 nm nanoparticle particles(SFPZ) coated with 17 pMHCs have higher agonistic activity than PFM(20-25 nm) particles coated with 53 pMHCs per NP. This confirms thatwith both class I-pMHC Nps and class II-pMHC-Nps it is not the absolutevalency of pMHC but rather the pMHC density that matters. Asdemonstrated by the increased pMHC-NP-induced secretion of IFNγ by CD4+T cells as a function of pMHC density.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”; and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anexcipient” includes a plurality of excipients.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein the followingterms have the following meanings.

As used herein, the term “comprising” or “comprises” is intended to meanthat the compositions and methods include the recited elements, but notexcluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention, such ascompositions for treating or preventing multiple sclerosis. “Consistingof” shall mean excluding more than trace elements of other ingredientsand substantial method steps. Embodiments defined by each of thesetransition terms are within the scope of this invention.

By “biocompatible”, it is meant that the components of the deliverysystem will not cause tissue injury or injury to the human biologicalsystem. To impart biocompatibility, polymers and excipients that havehad history of safe use in humans or with GRAS (Generally Accepted AsSafe) status, will be used preferentially. By biocompatibility, it ismeant that the ingredients and excipients used in the composition willultimately be “bioabsorbed” or cleared by the body with no adverseeffects to the body. For a composition to be biocompatible, and beregarded as non-toxic, it must not cause toxicity to cells. Similarly,the term “bioabsorbable” refers to nanoparticles made from materialswhich undergo bioabsorption in vivo over a period of time such that longterm accumulation of the material in the patient is avoided. In apreferred embodiment, the biocompatible nanoparticle is bioabsorbed overa period of less than 2 years, preferably less than 1 year and even morepreferably less than 6 months. The rate of bioabsorption is related tothe size of the particle, the material used, and other factors wellrecognized by the skilled artisan. A mixture of bioabsorbable,biocompatible materials can be used to form the nanoparticles used inthis invention. In one embodiment, iron oxide and a biocompatible,bioabsorbable polymer can be combined. For example, iron oxide and PGLAcan be combined to form a nanoparticle.

An antigen-MHC-nanoparticle complex refers to presentation of a peptide,carbohydrate, lipid, or other antigenic segment, fragment, or epitope ofan antigenic molecule or protein (i.e., self peptide or autoantigen) ona surface, such as a biocompatible biodegradable nanosphere. “Antigen”as used herein refers to all, part, fragment, or segment of a moleculethat can induce an immune response in a subject or an expansion ofanti-pathogenic cells.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, and concentration, including range, indicatesapproximations which may vary by (+) or (−) 10%, 5%, or 1%.

A “mimic” is an analog of a given ligand or peptide, wherein the analogis substantially similar to the ligand. “Substantially similar” meansthat the analog has a binding profile similar to the ligand except themimic has one or more functional groups or modifications thatcollectively accounts for less than about 50%, less than about 40%, lessthan about 30%, less than about 20%, less than about 10%, or less thanabout 5% of the molecular weight of the ligand.

Multiple sclerosis (MS) is also known as “disseminated sclerosis,”“encephalomyelitis disseminate,” or “allergic encephalomyelitis.” MS isan inflammatory disease in which the fatty myelin sheaths around theaxons of the brain and spinal cord are damaged, leading to demyelinationand scarring as well as a broad spectrum of signs and symptoms. Multiplesclerosis-related disorders include, for example, neuromyelitis optica(NMO), uveitis, neuropathis pain, and the like.

“Myelin Oligodendrocyte Glycoprotein” (MOG) is a glycoprotein believedto be important in the process of myelinization of nerves in the centralnervous system (CNS). In humans this protein is encoded by the MOG gene.It is speculated to serve as a necessary “adhesion molecule” to providestructural integrity to the myelin sheath and is known to develop lateon the oligodendrocyte. The GenBank accession numbers NM_001008228.2 andNP_001008229.1 represent the mRNA and protein sequence, respectively, ofthe MOG gene. The sequence associated with each of these GenBankaccession numbers is incorporated by reference for all purposes.

The term “anti-pathogenic autoreactive T cell” refers to a T cell withanti-pathogenic properties (i.e. T cells that counteract MS). These Tcells can include anti-inflammatory T cells, effector T cells, memory Tcells, low-avidity T cells, T helper cells, autoregulatory T cells,cytotoxic T cells, natural killer T cells, CD4+ T cells, CD8+ T cellsand the like.

The term “anti-inflammatory T cell” refers to a T cell that promotes ananti-inflammatory response. The anti-inflammatory function of the T cellmay be accomplished through production and/or secretion ofanti-inflammatory proteins, cytokines, chemokines, and the like.Anti-inflammatory proteins are also intended to encompassanti-proliferative signals that suppress immune responses.Anti-inflammatory proteins include IL-4, IL-10, IL-13, IFN-α, TGF-β,IL-1ra, G-CSF, and soluble receptors for TNF and IL-6. In certainembodiments, administration of the antigen-MHC nanoparticle complexleads to expansion and or increased induction of anti-inflammatory Tcells effective for treating multiple sclerosis. Accordingly, aspects ofthe disclosure relate to methods for treating, in a patient,inflammation associated with MS, the method comprising, consistingessentially of or yet further consisting of administering to thatpatient an antigen-MHC-nanoparticle complex, wherein the antigen is amultiple sclerosis-related antigen.

The term “IL-10” or “Interleukin-10” refers to a cytokine encoded by theIL-10 gene. The IL-10 sequence is represented by the GenBank AccessionNo.: NM_000572.2 (mRNA) and NP_000563.1 (protein).

The term “TGF-β” or “Transforming growth factor beta” refers to aprotein that can have an anti-inflammatory effect. TGF-β is a secretedprotein that exists in at least three isoforms called TGF-β1, TGF-β2 andTGF-β3. It was also the original name for TGF-β1, which was the foundingmember of this family. The TGF-β family is part of a superfamily ofproteins known as the transforming growth factor beta superfamily, whichincludes inhibins, activin, anti-millerian hormone, bone morphogeneticprotein, decapentaplegic and Vg-1.

A “an effective amount” is an amount sufficient to achieve the intendedpurpose, non-limiting examples of such include: initiation of the immuneresponse, modulation of the immune response, suppression of aninflammatory response and modulation of T cell activity or T cellpopulations. In one aspect, the effective amount is one that-functionsto achieve a stated therapeutic purpose, e.g., a therapeuticallyeffective amount. As described herein in detail, the effective amount,or dosage, depends on the purpose and the composition, component and canbe determined according to the present disclosure.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

By “nanosphere,” “NP,” or “nanoparticle” herein is meant a smalldiscrete particle that is administered singularly or pluraly to asubject, cell specimen or tissue specimen as appropriate. In certainembodiments, the nanoparticles are substantially spherical in shape. Incertain embodiments, the nanoparticle is not a liposome or viralparticle. In further embodiments, the nanoparticle is solid. The term“substantially spherical,” as used herein, means that the shape of theparticles does not deviate from a sphere by more than about 10%. Variousknown antigen or peptide complexes of the invention may be applied tothe particles. The nanoparticles of this invention range in size fromabout 1 nm to about 1 μm and, preferably, from about 1 nm to about 100nm and in some aspects refers to the average or median diameter of aplurality of nanoparticles when a plurality of nanoparticles areintended. Smaller nanosize particles can be obtained, for example, bythe process of fractionation whereby the larger particles are allowed tosettle in an aqueous solution. The upper portion of the solution is thenrecovered by methods known to those of skill in the art. This upperportion is enriched in smaller size particles. The process can berepeated until a desired average size is generated.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used herein the phrase “immune response” or its equivalent“immunological response” refers to the development of a cell-mediatedresponse (mediated by antigen-specific T cells or their secretionproducts). A cellular immune response is elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules, to activate antigen-specific CD4⁺ T helper cells and/or CD8+cytotoxic T cells. The response may also involve activation of othercomponents.

The terms “inflammatory response” and “inflammation” as used hereinindicate the complex biological response of vascular tissues of anindividual to harmful stimuli, such as pathogens, damaged cells, orirritants, and includes secretion of cytokines and more particularly ofpro-inflammatory cytokines, i.e. cytokines which are producedpredominantly by activated immune cells and are involved in theamplification of inflammatory reactions. Exemplary pro-inflammatorycytokines include but are not limited to IL-1, IL-6, TNF-a, IL-17, IL21,IL23, and TGF-β. Exemplary inflammations include acute inflammation andchronic inflammation. Acute inflammation indicates a short-term processcharacterized by the classic signs of inflammation (swelling, redness,pain, heat, and loss of function) due to the infiltration of the tissuesby plasma and leukocytes. An acute inflammation typically occurs as longas the injurious stimulus is present and ceases once the stimulus hasbeen removed, broken down, or walled off by scarring (fibrosis). Chronicinflammation indicates a condition characterized by concurrent activeinflammation, tissue destruction, and attempts at repair. Chronicinflammation is not characterized by the classic signs of acuteinflammation listed above. Instead, chronically inflamed tissue ischaracterized by the infiltration of mononuclear immune cells(monocytes, macrophages, lymphocytes, and plasma cells), tissuedestruction, and attempts at healing, which include angiogenesis andfibrosis. An inflammation can be inhibited in the sense of the presentdisclosure by affecting and in particular inhibiting anyone of theevents that form the complex biological response associated with aninflammation in an individual.

The terms “epitope” and “antigenic determinant” are used interchangeablyto refer to a site on an antigen to which B and/or T cells respond orrecognize. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Glenn E. Morris, Epitope Mapping Protocols (1996). T-cells recognizecontinuous epitopes of about nine amino acids for CD8 cells or about13-15 amino acids for CD4 cells. T cells that recognize the epitope canbe identified by in vitro assays that measure antigen-dependentproliferation, as determined by ³H-thymidine incorporation by primed Tcells in response to an epitope (Burke et al., J. Inf. Dis.,170:1110-1119, 1994), by antigen-dependent killing (cytotoxic Tlymphocyte assay, Tigges et al., J. Immunol., 156(10):3901-3910, 1996)or by cytokine secretion. The presence of a cell-mediated immunologicalresponse can be determined by proliferation assays (CD4⁺ T cells) or CTL(cytotoxic T lymphocyte) assays.

Optionally, an antigen or preferably an epitope of an antigen, can bechemically conjugated to, or expressed as, a fusion protein with otherproteins, such as MHC and MHC related proteins.

As used herein, the terms “patient” and “subject” are used synonymouslyand refer to a mammal. In some embodiments the patient is a human. Inother embodiments the patient is a mammal commonly used in a laboratorysuch as a mouse, rat, simian, canine, feline, bovine, equine, or ovine.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule that either is recombinant or has been isolatedfree of total genomic nucleic acid. Included within the term“polynucleotide” are oligonucleotides (nucleic acids 100 residues orless in length), recombinant vectors, including, for example, plasmids,cosmids, phage, viruses, and the like. Polynucleotides include, incertain aspects, regulatory sequences, isolated substantially away fromtheir naturally occurring genes or protein encoding sequences.Polynucleotides may be RNA, DNA, analogs thereof, or a combinationthereof. A nucleic acid encoding all or part of a polypeptide maycontain a contiguous nucleic acid sequence encoding all or a portion ofsuch a polypeptide of the following lengths: 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, ormore nucleotides, nucleosides, or base pairs. It also is contemplatedthat a particular polypeptide from a given species may be encoded bynucleic acids containing natural variations that having slightlydifferent nucleic acid sequences but, nonetheless, encode the same orsubstantially similar protein, polypeptide, or peptide.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

The term “isolated” or “recombinant” as used herein with respect tonucleic acids, such as DNA or RNA, refers to molecules separated fromother DNAs or RNAs, respectively that are present in the natural sourceof the macromolecule as well as polypeptides. The term “isolated orrecombinant nucleic acid” is meant to include nucleic acid fragmentswhich are not naturally occurring as fragments and would not be found inthe natural state. The term “isolated” is also used herein to refer topolynucleotides, polypeptides and proteins that are isolated from othercellular proteins and is meant to encompass both purified andrecombinant polypeptides. In other embodiments, the term “isolated orrecombinant” means separated from constituents, cellular and otherwise,in which the cell, tissue, polynucleotide, peptide, polypeptide,protein, antibody or fragment(s) thereof, which are normally associatedin nature. For example, an isolated cell is a cell that is separatedfrom tissue or cells of dissimilar phenotype or genotype. An isolatedpolynucleotide is separated from the 3′ and 5′ contiguous nucleotideswith which it is normally associated in its native or naturalenvironment, e.g., on the chromosome. As is apparent to those of skillin the art, a non-naturally occurring polynucleotide, peptide,polypeptide, protein, antibody or fragment(s) thereof, does not require“isolation” to distinguish it from its naturally occurring counterpart.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) having a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. The alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in Current Protocols in MolecularBiology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table7.7.1. Preferably, default parameters are used for alignment. Apreferred alignment program is BLAST, using default parameters. Inparticular, preferred programs are BLASTN and BLASTP, using thefollowing default parameters: Genetic code=standard; filter=none;strand=both; cutoff⁼60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

It is to be inferred without explicit recitation and unless otherwiseintended, that when the present invention relates to a polypeptide,protein, polynucleotide or antibody, an equivalent or a biologicallyequivalent of such is intended within the scope of this invention. Asused herein, the term “biological equivalent thereof” is intended to besynonymous with “equivalent thereof” when referring to a referenceprotein, antibody, fragment, polypeptide or nucleic acid, intends thosehaving minimal homology while still maintaining desired structure orfunctionality. Unless specifically recited herein, it is contemplatedthat any polynucleotide, polypeptide or protein mentioned herein alsoincludes equivalents thereof. In one aspect, an equivalentpolynucleotide is one that hybridizes under stringent conditions to thepolynucleotide or complement of the polynucleotide as described hereinfor use in the described methods. In another aspect, an equivalentantibody or antigen binding polypeptide intends one that binds with atleast 70%, or alternatively at least 75%, or alternatively at least 80%,or alternatively at least 85%, or alternatively at least 90%, oralternatively at least 95% affinity or higher affinity to a referenceantibody or antigen binding fragment. In another aspect, the equivalentthereof competes with the binding of the antibody or antigen bindingfragment to its antigen under a competitive ELISA assay. In anotheraspect, an equivalent intends at least about 80% homology or identityand alternatively, at least about 85%, or alternatively at least about90%, or alternatively at least about 95%, or alternatively 98% percenthomology or identity and exhibits substantially equivalent biologicalactivity to the reference protein, polypeptide or nucleic acid.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PC reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubationtemperatures of about 25° C. to about 37° C.; hybridization bufferconcentrations of about 6×SSC to about 10×SSC; formamide concentrationsof about 0% to about 25%; and wash solutions from about 4×SSC to about8×SSC. Examples of moderate hybridization conditions include: incubationtemperatures of about 40° C. to about 50° C.; buffer concentrations ofabout 9×SSC to about 2×SSC; formamide concentrations of about 30% toabout 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples ofhigh stringency conditions include: incubation temperatures of about 55°C. to about 68° C.; buffer concentrations of about 1×SSC to about0.1×SSC; formamide concentrations of about 55% to about 75%; and washsolutions of about 1×SSC, 0.1×SSC, or deionized water. In general,hybridization incubation times are from 5 minutes to 24 hours, with 1,2, or more washing steps, and wash incubation times are about 1, 2, or15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It isunderstood that equivalents of SSC using other buffer systems can beemployed.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous”sequence shares lessthan 40% identity, or alternatively less than 25% identity, with one ofthe sequences of the present invention.

“Homology” or “identity” or “similarity” can also refer to two nucleicacid molecules that hybridize under stringent conditions.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be therapeutic in terms of a partial or completecure for a disorder and/or adverse effect attributable to the disorder.In one aspect, treatment indicates a reduction in the signs of thedisease using an established scale.

As used herein, the term “multiple sclerosis-related disorder” intends adisorder that co-presents with a susceptibility to MS or with MS.Non-limiting examples of such include neuromyelitis optica (NMO),uveitis, neuropathis pain clerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata systemic sclerosis, spino-optical MS, primaryprogressive MS (PPMS), and relapsing remitting MS (RRMS), progressivesystemic sclerosis, and ataxic sclerosis, IGRP, which is encoded by agene (located on chromosome 2q28-32 that overlaps a T1D susceptibilitylocus, IDDM7 (2q31), has also been recently identified as a beta-cellautoantigen of potential relevance in human T1D. Two HLA-A*0201-bindingepitopes of human IGRP (hIGRP₂₂₈₋₂₃₆ and hIGRP₂₆₅₋₂₇₃) are recognized byislet-associated CD8+ cells from murine MIIC class I-deficient NOD miceexpressing an HLA-A*0201 transgene. IGRP₂₀₆₋₂₁₄ comprises the antigenicpeptide VYLKTNVFL (SEQ ID NO: 19).

To prevent intends to prevent a disorder or effect in vitro or in vivoin a system or subject that is predisposed to the disorder or effect.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant. In certain embodiments, thecomposition does not contain an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see below Table).

Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCU CysteineCys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACI Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

As used herein, a “protein” or “polypeptide” or “peptide” refers to amolecule comprising at least five amino acid residues.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

Descriptive Embodiments

This disclosure is based on the discovery that nanoparticles coupled toMS-relevant antigen-MHC complexes reduce MS or encephalomyelitis (EAE)symptoms (Example 1).

II. METHODS

The methods as described herein comprise, or alternatively consistessentially of, or yet further consist of the administration of aneffective amount of an antigen-MHC-nanoparticle complex to a cell,tissue or subject for the purpose of: (1) expanding and/or developingpopulations of anti-pathogenic (or anti-MS) autoreactive T-cells; and/or(2) treating or preventing multiple sclerosis or a multiplesclerosis-related disorder in a patient with multiple sclerosis or amultiple sclerosis-related disorder or in a patient susceptible tomultiple sclerosis or a multiple sclerosis-related disorder, in oneaspect without compromising systemic immunity. The antigen used in thecomplex is a multiple sclerosis-related antigen. Methods to determineand monitor the therapy are known in the art and briefly describedherein. When delivered in vitro, administration is by contacting thecomposition with the tissue or cell by any appropriate method, e.g., byadministration to cell or tissue culture medium and is useful as ascreen to determine if the therapy is appropriate for an individual orto screen for alternative therapies to be used as a substitute or incombination with the disclosed compositions. When administered in vivo,administration is by systemic or local administration. In vivo, themethods can be practiced on a non-human animal to screen alternativetherapies to be used as a substitute or in combination with thedisclosed compositions prior to human administration. In a human ornon-human mammal, they are also useful to treat the disease or disorder.

The methods require administration of an effective amount of a complexcomprising, consisting essentially of or yet further consisting of, ananoparticle; a MHC protein; and a multiple sclerosis-related antigen.

The MHC of the antigen-MHC-nanoparticle complex can be MHC I, MHC II, ornon-classical MHC. MHC proteins are described herein. In one embodiment,the MHC of the antigen-MHC-nanoparticle complex is a MHC class I. Inanother embodiment, the MHC is a MHC class II. In other embodiments, theMHC component of the antigen-MHC-nanoparticle complex is MHC class II ora non-classical MHC molecule as described herein. In one aspect, theantigen comprises, or alternatively consists essentially of, or yetfurther consists of the polypeptide GWYRSPFSRVVH (SEQ ID NO: 1) or anequivalent of SEQ ID NO: 1. Additional antigens that can be used in thisinvention comprise polypeptides comprising, or alternatively consistingessentially of, or yet further consisting of the polypeptides of thegroup: MOG₃₅₋₅₅, MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 4); MOG₃₆₋₅₅,EVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 5); MAG₂₈₇₋₂₉₅, SLLLELEEV (SEQ ID NO:6); MAG₅₀₉₋₅₁₇, LMWAKIGPV (SEQ ID NO: 7); MAG₅₅₆₋₅₆₄, VLFSSDFRI (SEQ IDNO: 8); MBPI₁₁₀₋₁₁₈, SLSRFSWGA (SEQ ID NO: 9); MOG₁₁₄₋₁₂₂, KVEDPFYWV(SEQ ID NO: 10); MOG₁₆₆₋₁₇₅, RTFDPHFLRV (SEQ ID NO: 11); MOG₁₇₂₋₁₈₀,FLRVPCWKI (SEQ ID NO: 12); MOG₁₇₉₋₁₈₈, KITLFVIVPV (SEQ ID NO: 13);MOG₁₈₈₋₁₉₆, VLGPLVALI (SEQ ID NO: 14); MOG₈₁₋₁₈₉, TLFVIVPVL (SEQ ID NO:15); MOG₂₀₅₋₂₁₄, RLAGQFLEEL (SEQ ID NO: 16); PLP₈₀₋₈₈, FLYGALLLA (SEQ IDNO: 17), or an equivalent of each thereof, or combinations thereof.

The size of the nanoparticle can range from about 1 nm to about 1 μm. Incertain embodiments, the nanoparticle is less than about 1 μm indiameter. In other embodiments, the nanoparticle is less than about 500nm, less than about 400 nm, less than about 300 nm, less than about 200nm, less than about 100 nm, or less than about 50 nm in diameter. Infurther embodiments, the nanoparticle is from about 1 nm to about 10 nm,15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm in diameter.In specific embodiments, the nanoparticle is from about 1 nm to about100 nm, about 1 nm to about 50 nm, about 1 nm to about 20 nm, or about 5nm to about 20 nm.

The size of the complex can range from about 5 nm to about 1 km. Incertain embodiments, the complex is less than about 1 μm oralternatively less than 100 nm in diameter. In other embodiments, thecomplex is less than about 500 nm, less than about 400 nm, less thanabout 300 nm, less than about 200 nm, less than about 100 nm, or lessthan about 50 nm in diameter. In further embodiments, the complex isfrom about 10 nm to about 50 nm, or about 20 nm to about 75 nm, or about25 nm to about 60 nm, or from about 30 nm to about 60 nm, or in oneaspect about 55 nm.

Applicant has discovered that the density of the antigen-MHC complexeson the nanoparticle contributes to the therapeutic benefit. Thus, asdisclosed herein the antigen-MHC nanoparticle complex can have a defineddensity in the range of from about 0.05 MHC molecules per 100 nm² ofsurface area of the nanoparticle, assuming at least 2 MHC, oralternatively at least 8, or alternatively at least 9, or alternativelyat least 10, or alternatively at least 11, or alternatively at least 12,MHC complexed to the nanoparticle. In one aspect the complex has andensity of MHC from about 0.01 MHC per 100 nm² (0.05 MHC/100 nm²) toabout 30 MHC/100 nm², or alternatively from 0.1 MHC/100 nm² to about 25MHC/100 nm², or alternatively from about 0.3 MHC/100 nm² to about 25MHC/100 nm², or alternatively from about 0.4 MHC/100 nm² to about 25MHC/100 nm², or alternatively from about 0.5 MHC/100 nm² to about 20MHC/100 nm², or alternatively from about, or alternatively from 0.6MHC/100 nm² to about 20 MHC/100 nm², or alternatively from about 1.0MHC/100 nm² to about 20 MHC/100 nm², or alternatively from about 5.0MHC/100 nm² to about 20 MHC/100 nm², or alternatively from about 10.0MHC/100 nm² to about 20 MHC/100 nm², or alternatively from about 15MHC/100 nm² to about 20 MHC/100 nm², or alternatively at least about0.5, or alternatively at least about 1.0, or alternatively at leastabout 5.0, or alternatively at least about 10.0, or alternatively atleast about 15.0 MHC/100 nm². In one aspect, when 9 or at least 9 MHCare complexed to a nanoparticle, the density range is from about 0.3MHC/100 nm² to about 20 MHC/100 nm².

In one of its method aspects, there is provided a method foraccumulating anti-inflammatory T cells in a patient in need thereof. Ina further embodiment, the T cell is a CD4+ or CD8+ T cell. In a relatedembodiment, the T cell secretes IL-10 or TGFβ. The method comprises,consists essentially of, or yet further consists of administering to apatient in need thereof on effective amount of the antigen-MHCnanoparticle complex as described herein.

In one embodiment, the methods described herein are for treating amultiple sclerosis-related disorder. The method comprises, consistsessentially of, or yet further consists of administering to a patient inneed thereof on effective amount of the antigen-MHC nanoparticle complexas described herein. In a related embodiment, the multiplesclerosis-related disorder is selected from the group consisting ofneuromyelitis optica (NMO), uveitis, and neuropathis pain.

Details regarding modes of administration in vitro and in vivo aredescribed within.

III. ANTIGEN-MHC-NANOPARTICLE COMPLEXES

Certain aspects relate to processes for producing MS antigen-specificmedicaments that specifically treat MS without compromising systemicimmunity. Example 2 describes the production of antigen-MHC-nanoparticlecomplexes. Antigen-MHC-nanoparticle complexes useful in this inventioncomprise a MS relevant antigen.

A. Polypeptides and Polynucleotides

Further aspects relate to an isolated or purified polypeptidecomprising, or consisting essentially of, or yet further consisting of,the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at leastabout 80% sequence identity, or alternatively at least 85%, oralternatively at least 90%, or alternatively at least 95%, oralternatively at least 98% sequence identity to SEQ ID NO: 1, orpolypeptides encoded by polynucleotides having at about 80% sequenceidentity, or alternatively at least 85%, or alternatively at least 90%,or alternatively at least 95%, or alternatively at least 98% sequenceidentity to the polynucleotide encoding SEQ ID NO: 1 or its complement,or a polypeptide encoded by a polynucleotide that hybridizes underconditions of moderate to high stringency to a polynucleotide encodingSEQ ID NO: 1 or its complement. Also provided are isolated and purifiedpolynucleotides encoding the polypeptide corresponding to SEQ ID NO: 1,at least about 80% sequence identify to SEQ ID NO: 1, or alternativelyat least 85%, or alternatively at least 90%, or alternatively at least95%, or alternatively at least 98% sequence identity to SEQ ID NO: 1 oran equivalent, or a polynucleotide that hybridizes under stringentconditions to the polynucleotide, its equivalent or its complement andisolated or purified polypeptides encoded by these polynucleotides. Thepolypeptides and polynucleotides can be combined with non-naturallyoccurring substances with which they are not associated with in nature,e.g., carriers, pharmaceutically acceptable carriers, vectors and MHCmolecules, nanoparticles as known in the art and as described herein.

Antigens, including segments, fragments and other molecules derived froman antigenic species, including but not limited to peptides,carbohydrates, lipids or other molecules presented by classical andnon-classical MHC molecules of the invention are typically complexed oroperatively coupled to a MHC molecule or derivative thereof. Antigenrecognition by T lymphocytes is major histocompatibility complex(MHC)-restricted. A given T lymphocyte will recognize an antigen onlywhen it is bound to a particular MHC molecule. In general, T lymphocytesare stimulated only in the presence of self-MHC molecules, and antigenis recognized as fragments of the antigen bound to self MHC molecules.MHC restriction defines T lymphocyte specificity in terms of the antigenrecognized and in terms of the MHC molecule that binds its antigenicfragment(s). In particular aspects certain antigens will be paired withcertain MHC molecules or polypeptides derived there from.

The term “operatively coupled” or “coated” as used herein, refers to asituation where individual polypeptide (e.g., MHC) and antigenic (e.g.,peptide) components are combined to form the active complex prior tobinding at the target site, for example, an immune cell. This includesthe situation where the individual polypeptide complex components aresynthesized or recombinantly expressed and subsequently isolated andcombined to form a complex, in vitro, prior to administration to asubject; the situation where a chimeric or fusion polypeptide (i.e.,each discrete protein component of the complex is contained in a singlepolypeptide chain) is synthesized or recombinantly expressed as anintact complex. Typically, polypeptide complexes are added to thenanoparticles to yield nanoparticles with adsorbed or coupledpolypeptide complexes having a ratio of number of molecules:number ofnanoparticle ratios from about, at least about or at most about 0.1,0.5, 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600,700, 800, 900, 1000, 1500 or more to: 1, more typically 0.1:1, 1:1 to50:1 or 300:1. The polypeptide content of the nanoparticles can bedetermined using standard techniques.

B. MHC Molecules

Intracellular and extracellular antigens present quite differentchallenges to the immune system, both in terms of recognition and ofappropriate response. Presentation of antigens to T cells is mediated bytwo distinct classes of molecules MHC class I (MHC-I) and MHC class II(MHC-II) (also identified as “pMHC” herein), which utilize distinctantigen processing pathways. Peptides derived from intracellularantigens are presented to CD8⁺ T cells by MHC class I molecules, whichare expressed on virtually all cells, while extracellularantigen-derived peptides are presented to CD4⁺ T cells by MHC-IImolecules. However, there are certain exceptions to this dichotomy.Several studies have shown that peptides generated from endocytosedparticulate or soluble proteins are presented on MHC-I molecules inmacrophages as well as in dendritic cells. In certain embodiments of theinvention, a particular antigen is identified and presented in theantigen-MHC-nanoparticle complex in the context of an appropriate MHCclass I or II polypeptide. In certain aspects, the genetic makeup of asubject may be assessed to determine which MHC polypeptide is to be usedfor a particular patient and a particular set of peptides. In certainembodiments, the MHC class 1 component comprises all or part of a HLA-A,HLA-B, HLA-C, HLA-E, HLA-F, HLA-G or CD-1 molecule. In embodimentswherein the MHC component is a MHC class II component, the MHC class IIcomponent can comprise all or a part of a HLA-DR, HLA-DQ, or HLA-DP.

Non-classical MHC molecules are also contemplated for use in MHCcomplexes of the invention. Non-classical MHC molecules arenon-polymorphic, conserved among species, and possess narrow, deep,hydrophobic ligand binding pockets. These binding pockets are capable ofpresenting glycolipids and phospholipids to Natural Killer T (NKT) cellsor certain subsets of CD8+ T-cells such as Qa1 or HLA-E-restricted CD8+T-cells. NKT cells represent a unique lymphocyte population thatco-express NK cell markers and a semi-invariant T cell receptor (TCR).They are implicated in the regulation of immune responses associatedwith a broad range of diseases.

C. Antigenic Components

Certain aspects of the invention include methods and compositionsconcerning antigenic compositions including segments, fragments, orepitopes of polypeptides, peptides, nucleic acids, carbohydrates, lipidsand other molecules that provoke or induce an antigenic response,generally referred to as antigens. In particular, antigenic segments orfragments of antigenic determinants, which lead to the destruction of acell via an autoimmune response, can be identified and used in making anantigen-MHC-nanoparticle complex described herein. Embodiments of theinvention include compositions and methods for the modulation of animmune response in a cell or tissue of the body.

Polypeptides and peptides of the invention may be modified by variousamino acid deletions, insertions, and/or substitutions. In particularembodiments, modified polypeptides and/or peptides are capable ofmodulating an immune response in a subject. In some embodiments, awild-type version of a protein or peptide are employed, however, in manyembodiments of the invention, a modified protein or polypeptide isemployed to generate an antigen-MHC-nanoparticle complex. Anantigen-MHC-nanoparticle complex can be used to generate ananti-inflammatory immune response, to modify the T cell population ofthe immune system (i.e., re-educate the immune system), and/or fosterthe recruitment and accumulation of anti-inflammatory T cells to aparticular tissue. The terms described above may be used interchangeablyherein. A “modified protein” or “modified polypeptide” or “modifiedpeptide” refers to a protein or polypeptide whose chemical structure,particularly its amino acid sequence, is altered with respect to thewild-type protein or polypeptide. In some embodiments, a modifiedprotein or polypeptide or peptide has at least one modified activity orfunction (recognizing that proteins or polypeptides or peptides may havemultiple activities or functions). It is specifically contemplated thata modified protein or polypeptide or peptide may be altered with respectto one activity or function yet retains a wild-type activity or functionin other respects, such as immunogenicity or ability to interact withother cells of the immune system when in the context of anMHC-nanoparticle complex.

Antigens of the invention include antigens related to multiplesclerosis. Such antigens include, for example, those disclosed in USPat. App. No: 2012-0077686, and antigens derived from myelin basicprotein, myelin associated glycoprotein, myelin oligodendrocyte protein,proteolipid protein, oligodendrocyte myelin oligoprotein, myelinassociated oligodendrocyte basic protein, oligodendrocyte specificprotein, heat shock proteins, oligodendrocyte specific proteins NOGO A,glycoprotein Po, peripheral myelin protein 22, and 2′3′-cyclicnucleotide 3′-phosphodiesterase. In certain embodiments, the antigen isderived from antigen is derived from Myelin Oligodendrocyte Glycoprotein(MOG). In a related embodiment, the antigen corresponds to a peptidehaving at least 80% identity to a peptide comprising the sequence of SEQID NO: 1 or a polypeptide encoded by a polynucleotide that hybridizesunder conditions of moderate to high stringency to a polynucleotide thatencodes a sequence of SEQ ID NO: 1 or one having at least about 80%sequence identity to a sequence of SEQ ID NO: 1 or a complement thereof.

In certain embodiments, the size of a protein or polypeptide (wild-typeor modified), including any complex of a protein or peptide of interestand in particular a MHC-peptide fusion, may comprise, but is not limitedto 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800,825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500,1750, 2000, 2250, 2500 amino molecules or greater, including any rangeor value derivable therein, or derivative thereof. In certain aspects,5, 6, 7, 8, 9, 10 or more contiguous amino acids, including derivativesthereof, and fragments of an antigen, such as those amino acid sequencesdisclosed and referenced herein, can be used as antigens. It iscontemplated that polypeptides may be mutated by truncation, renderingthem shorter than their corresponding wild-type form, but also theymight be altered by fusing or conjugating a heterologous proteinsequence with a particular function (e.g., for presentation as a proteincomplex, for enhanced immunogenicity, etc.).

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including (i) the expression of proteins,polypeptides, or peptides through standard molecular biologicaltechniques, (ii) the isolation of proteinaceous compounds from naturalsources, or (iii) the chemical synthesis of proteinaceous materials. Thenucleotide as well as the protein, polypeptide, and peptide sequencesfor various genes have been previously disclosed, and may be found inthe recognized computerized databases. One such database is the NationalCenter for Biotechnology Information's GenBank and GenPept databases (onthe World Wide Web at ncbi.nlm.nih.gov/). The all or part of the codingregions for these genes may be amplified and/or expressed using thetechniques disclosed herein or as would be known to those of ordinaryskill in the art.

Amino acid sequence variants of autoantigenic epitopes and otherpolypeptides of these compositions can be substitutional, insertional,or deletion variants. A modification in a polypeptide of the inventionmay affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363,364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419,420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447,448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461,462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475,476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489,490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 Or morenon-contiguous or contiguous amino acids of a peptide or polypeptide, ascompared to wild-type.

Deletion variants typically lack one or more residues of the native orwild-type amino acid sequence. Individual residues can be deleted or anumber of contiguous amino acids can be deleted. A stop codon may beintroduced (by substitution or insertion) into an encoding nucleic acidsequence to generate a truncated protein. Insertional mutants typicallyinvolve the addition of material at a non-terminal point in thepolypeptide. This may include the insertion of one or more residues.Terminal additions, called fusion proteins, may also be generated.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of a polypeptide orpeptide is affected, such as avidity or affinity for a cellularreceptor(s). Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

Proteins of the invention may be recombinant, or synthesized in vitro.Alternatively, a recombinant protein may be isolated from bacteria orother host cell.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids, or 5′ or 3′ nucleic acid sequences, respectively, and yetstill be essentially as set forth in one of the sequences disclosedherein, so long as the sequence meets the criteria set forth above,including the maintenance of biological protein activity (e.g.,immunogenicity). The addition of terminal sequences particularly appliesto nucleic acid sequences that may, for example, include variousnon-coding sequences flanking either of the 5′ or 3′ portions of thecoding region.

It is contemplated that in compositions of the invention, there isbetween about 0.001 mg and about 10 mg of total protein per ml. Thus,the concentration of protein in a composition can be about, at leastabout or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 50, 100 μg/ml or mg/ml ormore (or any range derivable therein). Of this, about, at least about,or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may beantigen-MHC-nanoparticle complex.

The present invention contemplates the administration of anantigen-MHC-nanoparticle complex to effect a treatment against MS orand/or inflammation associated with MS.

In addition, U.S. Pat. No. 4,554,101 (Hopp), which is incorporatedherein by reference, teaches the identification and preparation ofepitopes from primary amino acid sequences on the basis ofhydrophilicity. Through the methods disclosed in Hopp, one of skill inthe art would be able to identify potential epitopes from within anamino acid sequence and confirm their immunogenicity. Numerousscientific publications have also been devoted to the prediction ofsecondary structure and to the identification of epitopes, from analysesof amino acid sequences (Chou & Fasman, Adv. Enzymol., 47:45-148, 1978;Chous and Fasman, Annu, Rev. Biochem., 47:251-276, 1978, Chou andFasman, Biochemistry, 13(2):211-222, 1974; Chau and Fasman,Biochemistry, 13(2):222-245, 1974, Chou and Fasman, Biophys. J.,26(3):385-399, 1979). Any of these may be used, if desired, tosupplement the teachings of Hopp in U.S. Pat. No. 4,554,101.

Molecules other than peptides can be used as antigens or antigenicfragments in complex with MHC molecules, such molecules include, but arenot limited to carbohydrates, lipids, small molecules, and the like.Carbohydrates are major components of the outer surface of a variety ofcells. Certain carbohydrates are characteristic of different stages ofdifferentiation and very often these carbohydrates are recognized byspecific antibodies. Expression of distinct carbohydrates can berestricted to specific cell types.

D. Substrates/Nanoparticles

In certain aspect, antigen/MHC complexes are operatively coupled to asubstrate which can be bound covalently or non-covently to the substrateA substrate can be in the form of a nanoparticle that optionallycomprises a biocompatible and/or bioabsorbable material. Accordingly, inone embodiment, the nanoparticle is biocompatible and/or bioabsorbable.A substrate can also be in the form of a nanoparticle such as thosedescribed previously in US Patent Pub. No.: 2009/0155292 which is hereinincorporated by reference in its entirety, which in one aspect is not aliposome Nanoparticles can have a structure of variable dimension andknown variously as a nanosphere, a nanoparticle or a biocompatiblebiodegradable nanosphere or a biocompatible biodegradable nanoparticle.Such particulate formulations containing an antigen/MHC complex can beformed by covalent or non-covalent coupling of the complex to thenanoparticle.

The nanoparticles typically consist of a substantially spherical coreand optionally one or more layers. The core may vary in size andcomposition. In addition to the core, the nanoparticle may have one ormore layers to provide functionalities appropriate for the applicationsof interest. The thicknesses of layers, if present, may vary dependingon the needs of the specific applications. For example, layers mayimpart useful optical properties.

Layers may also impart chemical or biological functionalities, referredto herein as chemically active or biologically active layers, and forthese functionalities the layer or layers may typically range inthickness from about 0.001 micrometers (1 nanometer) to about 10micrometers or more (depending on the desired nanoparticle diameter),these layers typically being applied on the outer surface of thenanoparticle.

The compositions of the core and layers may vary. Suitable materials forthe particles or the core include, but are not limited to polymers,ceramics, glasses, minerals, and the like. Examples include, but are notlimited to, standard and specialty glasses, silica, polystyrene,polyester, polycarbonate, acrylic polymers, polyacrylamide,polyacrylonitrile, polyamide, fluoropolymers, silicone, celluloses,silicon, metals (e.g., iron, gold, silver), minerals (e.g., ruby),nanoparticles (e.g., gold nanoparticles, colloidal particles, metaloxides, metal sulfides, metal selenides, and magnetic materials such asiron oxide), and composites thereof. The core could be of homogeneouscomposition, or a composite of two or more classes of material dependingon the properties desired. In certain aspects, metal nanoparticles willbe used. These metal particles or nanoparticles can be formed from Au,Pt, Pd, Cu, Ag, Co, Fe, Ni, Mn, Sm, Nd, Pr, Gd, Ti, Zr, Si, and In,precursors, their binary alloys, their ternary alloys and theirintermetallic compounds. See U.S. Pat. No. 6,712,997, which isincorporated herein by reference in its entirety. In certainembodiments, the compositions of the core and layers may vary providedthat the nanoparticles are biocompatible and bioabsorbable. The corecould be of homogeneous composition, or a composite of two or moreclasses of material depending on the properties desired. In certainaspects, metal nanospheres will be used. These metal nanoparticles canbe formed from Fe, Ca, Ga and the like. In certain embodiments, thenanoparticle comprises a core comprising metal or metal oxide.

As previously stated, the nanoparticle may, in addition to the core,include one or more layers. The nanoparticle may include a layerconsisting of a biodegradable sugar or other polymer. Examples ofbiodegradable layers include but are not limited to dextran;poly(ethylene glycol); poly(ethylene oxide); mannitol; poly(esters)based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL);poly(hydroxalkanoate)s of the PHB-PHV class; and other modifiedpoly(saccharides) such as starch, cellulose and chitosan. Additionally,the nanoparticle may include a layer with suitable surfaces forattaching chemical functionalities for chemical binding or couplingsites.

Layers can be produced on the nanoparticles in a variety of ways knownto those skilled in the art. Examples include sol-gel chemistrytechniques such as described in Iler, Chemistry of Silica, John Wiley &Sons, 1979; Brinker and Scherer, Sol-gel Science, Academic Press,(1990). Additional approaches to producing layers on nanoparticlesinclude surface chemistry and encapsulation techniques such as describedin Partch and Brown, J. Adhesion, 67:259-276, 1998; Pekarek et al.,Nature, 367:258, (1994); Hanprasopwattana, Langmuir, 12:3173-3179,(1996); Davies, Advanced Materials, 10:1264-1270, (1998); and referencestherein. Vapor deposition techniques may also be used; see for exampleGolman and Shinohara, Trends Chem. Engin., 6:1-6, (2000); and U.S. Pat.No. 6,387,498. Still other approaches include layer-by-layerself-assembly techniques such as described in Sukhorukov et al.,Polymers Adv. Tech., 9(10-11):759-767, (1998); Caruso et al.,Macromolecules, 32(7):2317-2328, (1998); Caruso et al., J. Amer. Chem.Soc., 121(25):6039-6046, (1999); U.S. Pat. No. 6,103,379 and referencescited therein.

Nanoparticles may be formed by contacting an aqueous phase containingthe antigen/MIIC/co-stimulatory molecule complex and a polymer and anonaqueous phase followed by evaporation of the nonaqueous phase tocause the coalescence of particles from the aqueous phase as taught inU.S. Pat. No. 4,589,330 or 4,818,542. Preferred polymers for suchpreparations are natural or synthetic copolymers or polymers selectedfrom the group consisting of gelatin agar, starch, arabinogalactan,albumin, collagen, polyglycolic acid, polylactic acid, glycolide-L(−)lactide poly(epsilon-caprolactone, poly(epsilon-caprolactone-CO-lacticacid), poly(epsilon-caprolactone-CO-glycolic acid), poly(β-hydroxybutyric acid), poly(ethylene oxide), polyethylene,poly(alkyl-2-cyanoacrylate), poly(hydroxyethyl methacrylate),polyamides, poly(amino acids), poly(2-hydroxyethyl DL-aspartamide),poly(ester urea), poly(L-phenylalanine/ethyleneglycol/1,6-diisocyanatohexane) and poly(methyl methacrylate).Particularly preferred polymers are polyesters, such as polyglycolicacid, polylactic acid, glycolide-L(−) lactide poly(epsilon-caprolactone,poly(epsilon-caprolactone-CO-lactic acid), andpoly(epsilon-caprolactone-CO-glycolic acid. Solvents useful fordissolving the polymer include: water, hexafluoroisopropanol,methylenechloride, tetrahydrofuran, hexane, benzene, orhexafluoroacetone sesquihydrate.

The size of the nanoparticle can range from about 1 nm to about 1 μm. Incertain embodiments, the nanoparticle is less than about 1 μm indiameter. In other embodiments, the nanoparticle is less than about 500nm, less than about 400 nm, less than about 300 nm, less than about 200nm, less than about 100 nm, or less than about 50 nm in diameter. Infurther embodiments, the nanoparticle is from about 1 nm to about 10 nm,15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm in diameter.In specific embodiments, the nanoparticle is from about 1 nm to about100 nm, about 1 nm to about 50 nm, about 1 nm to about 20 nm, or about 5nm to about 20 nm.

The size of the complex can range from about 5 nm to about 1 μm. Incertain embodiments, the complex is less than about 1 μm oralternatively less than 100 nm in diameter. In other embodiments, thecomplex is less than about 500 nm, less than about 400 nm, less thanabout 300 nm, less than about 200 nm, less than about 100 nm, or lessthan about 50 nm in diameter. In further embodiments, the complex isfrom about 10 nm to about 50 nm, or about 20 nm to about 75 nm, or about25 nm to about 60 nm, or from about 30 nm to about 60 nm, or in oneaspect about 55 nm.

E. Coupling Antigen-MHC Complex with the Nanoparticle

In order to couple the substrate or nanospheres to the antigen-MHCcomplexes the following techniques can be applied.

The binding can be generated by chemically modifying the substrate ornanoparticle which typically involves the generation of “functionalgroups” on the surface, said functional groups being capable of bindingto an antigen-MHC complex, and/or linking the optionally chemicallymodified surface of the substrate or nanoparticle with covalently ornon-covalently bonded so-called “linking molecules,” followed byreacting the antigen-MHC complex with the nanoparticles obtained.

The term “linking molecule” means a substance capable of linking withthe substrate or nanoparticle and also capable of linking to anantigen-MHC complex. In certain embodiments, the antigen-MHC complexesare coupled to the nanoparticle by a linker. Non-limiting examples ofsuitable linkers include dopamine (DPA)-polyethylene glycol (PEG)linkers such as DPA-PEG-NHS ester, DPA-PEG-orthopyridyl-disulfide (OPSS)and/or DPA-PEG-Azide. Other linkers include peptide linkers, ethyleneglycol, biotin, and strepdavidin.

The term “functional groups” as used herein before is not restricted toreactive chemical groups forming covalent bonds, but also includeschemical groups leading to an ionic interaction or hydrogen bonds withthe antigen-MHC complex. Moreover, it should be noted that a strictdistinction between “functional groups” generated at the surface andlinking molecules bearing “functional groups” is not possible, sincesometimes the modification of the surface requires the reaction ofsmaller linking molecules such as ethylene glycol with the nanospheresurface.

The functional groups or the linking molecules bearing them may beselected from amino groups, carbonic acid groups, thiols, thioethers,disulfides, guanidino, hydroxyl groups, amine groups, vicinal dioles,aldehydes, alpha-haloacetyl groups, mercury organyles, ester groups,acid halide, acid thioester, acid anhydride, isocyanates,isothiocyanates, sulfonic acid halides, imidoesters, diazoacetates,diazonium salts, 1,2-diketones, phosphonic acids, phosphoric acidesters, sulfonic acids, azolides, imidazoles, indoles, N-maleimides,alpha-beta-unsaturated carbonyl compounds, arylhalogenides or theirderivatives.

Non-limiting examples for other linking molecules with higher molecularweights are nucleic acid molecules, polymers, copolymers, polymerizablecoupling agents, silica, proteins, and chain-like molecules having asurface with the opposed polarity with respect to the substrate ornanoparticle. Nucleic acids can provide a link to affinity moleculescontaining themselves nucleic acid molecules, though with acomplementary sequence with respect to the linking molecule.

A specific example of a covalent linker includes poly(ethylene) glycol(PEG). The PEG linker may be a thiol-PEG-NH₂ linker.

In certain embodiments, the linker as described herein has a definedsize. In some embodiments, the linker is less that about 10 kD, lessthan about 5 kD, less than about 4.5 kD, less than about 4 kD, less thanabout 3.5 kD, less than about 3 kD, less than about 2.5 kD, less thanabout 2 kD, or less than about 1 kD. In further embodiments, the linkeris from about 0.5 kD to about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 kD.In yet further embodiments, the linker is from about 1 to about, 4.5, 4,3.5, 3, 2.5, 2, or 1.5 kD.

As examples for polymerizable coupling agents, diacetylene, styrenebutadiene, vinylacetate, acrylate, acrylamide, vinyl compounds, styrene,silicone oxide, boron oxide, phosphorous oxide, borates, pyrrole,polypyrrole and phosphates can be cited.

The surface of the substrate or nanoparticle can be chemically modified,for instance by the binding of phosphonic acid derivatives havingfunctional reactive groups. One example of these phosphonic acid orphosphonic acid ester derivates is imino-bis(methylenphosphono) carbonicacid which can be synthesized according to the “Mannich-Moedritzer”reaction. This binding reaction can be performed with substrate ornanosphere as directly obtained from the preparation process or after apre-treatment (for instance with trimethylsilyl bromide). In the firstcase the phosphonic acid (ester) derivative may for instance displacecomponents of the reaction medium which are still bound to the surface.This displacement can be enhanced at higher temperatures. Trimethylsilylbromide, on the other hand, is believed to dealkylate alkylgroup-containing phosphorous-based complexing agents, thereby creatingnew binding sites for the phosphonic acid (ester) derivative. Thephosphonic acid (ester) derivative, or linking molecules bound thereto,may display the same functional groups as given above. A further exampleof the surface treatment of the substrate or nanosphere involves heatingin a diole such as ethylene glycol. It should be noted that thistreatment may be redundant if the synthesis already proceeded in adiole. Under these circumstances the synthesis product directly obtainedis likely to show the necessary functional groups. This treatment ishowever applicable to substrate or nanoparticle that were produced in N-or P-containing complexing agents. If such substrate or particle aresubjected to an after-treatment with ethylene glycol, ingredients of thereaction medium (e.g. complexing agent) still binding to the surface canbe replaced by the diole and/or can be dealkylated.

It is also possible to replace N-containing complexing agents stillbound to the particle surface by primary amine derivatives having asecond functional group. The surface of the substrate or nanoparticlecan also be coated with silica. Silica allows a relatively simplechemical conjugation of organic molecules since silica easily reactswith organic linkers, such as triethoxysilane or chlorosilane. Thenanoparticle surface may also be coated by homo- or copolymers. Examplesfor polymerizable coupling agents are.N-(3-aminopropyl)-3-mercaptobenzamidine,3-(trimethoxysilyl)propylhydrazide and3-trimethoxysilyl)propylmaleimide. Other non-limiting examples ofpolymerizable coupling agents are mentioned herein. These couplingagents can be used singly or in combination depending on the type ofcopolymer to be generated as a coating.

Another surface modification technique that can be used with substratesor nanoparticles containing oxidic transition metal compounds isconversion of the oxidic transition metal compounds by chlorine gas ororganic chlorination agents to the corresponding oxychlorides. Theseoxychlorides are capable of reacting with nucleophiles, such as hydroxyor amino groups as often found in biomolecules. This technique allowsgenerating a direct conjugation with proteins, for instance-via theamino group of lysine side chains. The conjugation with proteins aftersurface modification with oxychlorides can also be effected by using abi-functional linker, such as maleimidopropionic acid hydrazide.

For non-covalent linking techniques, chain-type molecules having apolarity or charge opposite to that of the substrate or nanospheresurface are particularly suitable. Examples for linking molecules whichcan be non-covalently linked to core/shell nanospheres involve anionic,cationic or zwitter-ionic surfactants, acid or basic proteins,polyamines, polyamides, polysulfone or polycarboxylic acid. Thehydrophobic interaction between substrate or nanosphere and amphiphilicreagent having a functional reactive group can generate the necessarylink. In particular, chain-type molecules with amphiphilic character,such as phospholipids or derivatised polysaccharides, which can becrosslinked with each other, are useful. The absorption of thesemolecules on the surface can be achieved by coincubation. The bindingbetween affinity molecule and substrate or nanoparticle can also bebased on non-covalent, self-organising bonds. One example thereofinvolves simple detection probes with biotin as linking molecule andavidin- or strepdavidin-coupled molecules.

Protocols for coupling reactions of functional groups to biologicalmolecules can be found in the literature, for instance in “BioconjugateTechniques” (Greg T. Hermanson, Academic Press 1996). The biologicalmolecule (e.g., MHC molecule or derivative thereof) can be coupled tothe linking molecule, covalently or non-covalently, in line withstandard procedures of organic chemistry such as oxidation,halogenation, alkylation, acylation, addition, substitution oramidation. These methods for coupling the covalently or non-covalentlybound linking molecule can be applied prior to the coupling of thelinking molecule to the substrate or nanosphere or thereafter. Further,it is possible, by means of incubation, to effect a direct binding ofmolecules to correspondingly pre-treated substrate or nanoparticle (forinstance by trimethylsilyl bromide), which display a modified surfacedue to this pre-treatment (for instance a higher charge or polarsurface).

F. Protein Production

The present invention describes polypeptides, peptides, and proteins foruse in various embodiments of the present invention. For example,specific peptides and their complexes are assayed for their abilities toelicit or modulate an immune response. In specific embodiments, all orpart of the peptides or proteins of the invention can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, Solid Phase Peptide Synthesis, 2^(nd). Ed.,Pierce Chemical Co., (1984); Tam et al., J. Am. Chem. Soc., 105:6442,(1983); Merrifield, Science, 232(4748):341-347, (1986); and Barany andMerrifield, The Peptides, Gross and Meinhofer (Eds.), Academic Press,NY, 1-284, (1979), each incorporated herein by reference. Alternatively,recombinant DNA technology may be employed wherein a nucleotide sequencewhich encodes a peptide of the invention is inserted into an expressionvector, transformed or transfected into an appropriate host cell andcultivated under conditions suitable for expression.

One embodiment of the invention includes the use of gene transfer tocells, including microorganisms, for the production of proteins. Thegene for the protein of interest may be transferred into appropriatehost cells followed by culture of cells under the appropriateconditions. A nucleic acid encoding virtually any polypeptide may beemployed. The generation of recombinant expression vectors, and theelements included therein, are known to one skilled in the art and arebriefly discussed herein. Examples of mammalian host cell lines include,but are not limited to Vero and HeLa cells, other B- and T-cell lines,such as CEM, 721.221, H9, Jurkat, Raji, as well as cell lines of Chinesehamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. Inaddition, a host cell strain may be chosen that modulates the expressionof the inserted sequences, or that modifies and processes the geneproduct in the manner desired. Such modifications (e.g., glycosylation)and processing (e.g., cleavage) of protein products may be important forthe function of the protein. Different host cells have characteristicand specific mechanisms for the post-translational processing andmodification of proteins. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed

A number of selection systems may be used including, but not limited toHSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase,and adenine phosphoribosyltransferase genes, in tk-, hgprt- oraprt-cells, respectively. Also, anti-metabolite resistance can be usedas the basis of selection: for dhfr, which confers resistance totrimethoprim and methotrexate; gpt, which confers resistance tomycophenolic acid; neo, which confers resistance to the aminoglycosideG418; and hygro, which confers resistance to hygromycin.

G. Nucleic Acids

The present invention may include recombinant polynucleotides encodingthe proteins, polypeptides, peptides of the invention, such as, forexample, SEQ ID NO: 1 or 2.

In particular embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating nucleic acid sequencesthat encode an autoantigen and/or a MHC molecule. The term “recombinant”may be used in conjunction with a polypeptide or the name of a specificpolypeptide, and this generally refers to a polypeptide produced from anucleic acid molecule that has been manipulated in vitro or that is areplication product of such a molecule.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant nucleic acid protocol. In some cases, a nucleic acidsequence may encode a polypeptide sequence with additional heterologouscoding sequences, for example to allow for purification of thepolypeptide, transport, secretion, post-translational modification, orfor therapeutic benefits such as targeting or efficacy. A tag or otherheterologous polypeptide may be added to the modifiedpolypeptide-encoding sequence, wherein “heterologous” refers to apolypeptide that is not the same as the modified polypeptide.

IV. Pharmaceutical Compositions and Administration

Provided herein are pharmaceutical compositions useful for the treatmentof disease.

A. Pharmaceutical Compositions

The antigen-MHC nanoparticle complexes can be administered alone or incombination with a carrier, such as a pharmaceutically acceptablecarrier in a composition. Compositions of the invention may beconventionally administered parenterally, by injection, for example,intravenously, subcutaneously, or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude oral formulations. Oral formulations include such normallyemployed excipients such as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate and the like. These compositions take theform of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain about 10% to about 95% ofactive ingredient, preferably about 25% to about 70%. The preparation ofan aqueous composition that contains a antigen-MHC-nanoparticle complexthat modifies the subject's immune condition will be known to those ofskill in the art in light of the present disclosure. In certainembodiments, a composition may be inhaled (e.g., U.S. Pat. No.6,651,655, which is specifically incorporated by reference in itsentirety). In one embodiment, the antigen-MHC-nanoparticle complex isadministered systemically.

Typically, compositions of the invention are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective and immune modifying. The quantity to beadministered depends on the subject to be treated. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner. However, suitable dosage ranges are of the order often to several hundred nanograms or micrograms antigen-MHC-nanoparticlecomplex per administration. Suitable regimes for initial administrationand boosters are also variable, but are typified by an initialadministration followed by subsequent administrations.

In many instances, it will be desirable to have multiple administrationsof a peptide-MHC-nanoparticle complex, about, at most about or at leastabout 3, 4, 5, 6, 7, 8, 9, 10 or more. The administrations will normallyrange from 2 day to twelve week intervals, more usually from one to twoweek intervals. Periodic boosters at intervals of 0.5-5 years, usuallytwo years, may be desirable to maintain the condition of the immunesystem. The course of the administrations may be followed by assays forinflammatory immune responses and/or autoregulatory T cell activity.

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects of the present invention involveadministering an effective amount of a antigen-MHC-nanoparticle complexcomposition to a subject. Additionally, such compositions can beadministered in combination with modifiers of the immune system. Suchcompositions will generally be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal, or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The compositions may be formulated into a neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid poly(ethylene glycol), and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersion,and by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed bysterilization. Sterilization of the solution will be done in such a wayas to not diminish the therapeutic properties of theantigen-MHC-nanoparticle complex. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus anyadditional desired ingredient from a previously sterilized solutionthereof. One such method of sterilization of the solution is sterilefiltration, however, this invention is meant to include any method ofsterilization that does not significantly decrease the therapeuticproperties of the antigen-MHC-nanoparticle complexes. Methods ofsterilization that involve intense heat and pressure, such asautoclaving, may compromise the tertiary structure of the complex, thussignificantly decreasing the therapeutic properties of theantigen-MHC-nanoparticle complexes.

An effective amount of therapeutic composition is determined based onthe intended goal. The term “unit dose” or “dosage” refers to physicallydiscrete units suitable for use in a subject, each unit containing apredetermined quantity of the composition calculated to produce thedesired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the result and/or protection desired. Precise amounts of thecomposition also depend on the judgment of the practitioner and arepeculiar to each individual. Factors affecting dose include physical andclinical state of the subject, route of administration, intended goal oftreatment (alleviation of symptoms versus cure), and potency, stability,and toxicity of the particular composition. Upon formulation, solutionswill be administered in a manner compatible with the dosage formulationand in such amount as is therapeutically or prophylactically effective.The formulations are easily administered in a variety of dosage forms,such as the type of injectable solutions described above.

B. Combination Therapy

The compositions and related methods of the present invention,particularly administration of a antigen-MHC-nanoparticle complex, mayalso be used in combination with the administration of traditionaltherapies. These include, but are not limited to, Avonex (interferonbeta-1a), Betaseron (interferon beta-1b), Copaxone (glatiramer acetate),Novantrone (mitoxantrone), Rebif (interferon beta-1a), Tysabri(natalizumab), Gilenya (fingolimod), Glatiramer, steroids, Cytoxan,Imuran, Baclofen, deep brain stimulation, Ampyra (dalfampridine),acupuncture, and physical therapy.

When combination therapy is employed, various combinations may beemployed, for example antigen-MHC-nanoparticle complex administration is“A” and the additional agent is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A/ B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the peptide-MHC complex compositions of the presentinvention to a patient/subject will follow general protocols for theadministration of such compounds, taking into account the toxicity, ifany. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, suchas hydration, may be applied in combination with the described therapy.

C. In Vitro or Ex Vivo Administration

As used herein, the term in vitro administration refers to manipulationsperformed on cells removed from or outside of a subject, including, butnot limited to cells in culture. The term ex vivo administration refersto cells which have been manipulated in vitro, and are subsequentlyadministered to a subject. The term in vivo administration includes allmanipulations performed within a subject, including administrations.

In certain aspects of the present invention, the compositions may beadministered either in vitro, ex vivo, or in vivo. In certain in vitroembodiments, autologous T cells are incubated with compositions of thisinvention. The cells or tissue can then be used for in vitro analysis,or alternatively for ex vivo administration.

V. EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of embodiments andare exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1. pMHC Class II-NPs in Chronic EAE Autoimmune Disease Model

This example describes the use of nanoparticles coated with MS-relatedantigen-MHC complexes to treat EAE in an EAE mouse model. This noveltherapeutic approach can be used for systemic delivery of nanoparticlescoated with single MS-relevant peptideMHC complexes (pMHC) (i.e. onepMHC complex per disease). This discovery enables the rational design ofdisease-specific ‘nanovaccines’ capable of blunting autoimmunity withoutimpairing systemic immunity, a long sought-after goal in the therapy ofthese disorders. FIG. 1 shows that monospecific, MS-relevant pMHC classII coated nanovaccines can reverse established experimental allergicencephalomyelitis (EAE) in both C57BL/6 mice. This approach is alsoapplicable to human pMHC complexes known to be targeted by autoreactiveCD4+ T-cells in MS patients. Demonstration of clinical efficacy in thesepre-clinical models will pave the way for clinical trials in humans and,potentially, development of a cure for MS.

EAE requires the generation of autoreactive CD4+ cells along with thebreakdown of the bloodbrain barrier, which enables the recruitment ofencephalitogenic cells to the CNS. Immunization of C57BL/6 (B6) micewith pMOG₃₆₋₅₅ (200 μg) in CFA supplemented with 10 μg/ml Mycobacteriumtuberculosis s.c. (at the base of the tail) along with 300 ng ofPertussis toxin i.p., followed by another dose of Pertussis toxin on day2, induces a form of chronic EAE (>60 days) in all the animals that getsick (˜70% in our colony).

As shown in FIG. 1, pMHC class II-NP therapy (pMOG₃₈₋₄₉/IA^(b)-coatedNPs) reduces the severity of established EAE in C57BL/6 mice. B6 micewere immunized with pMOG₃₅₋₅₅ in CFA and treated with pertussis toxini.v. Mice were scored for signs of EAE using established criteria over a15-point scale. Affected mice were treated with two weekly doses of7.5-22.5 μg of pMOG₃₈₋₄₉-coated NPs, beginning 21 days afterimmunization. Importantly, this effect is associated with systemicexpansion of cognate autoreactive T-cells (FIG. 4). Furthermore, whereasthe spinal cords of untreated mice had significant demyelination anddense mononuclear cell infiltrates of the white matter (FIG. 5),pMHC-NP-treated mice had significantly less demyelination andmononuclear cell infiltrates (FIG. 6). FIGS. 7 and 8 show representativeexamples for the spinal cord edges (2 mice each). Here, again,pMHC-NP-treated mice have significantly less demyelination as well aslower mononuclear cell infiltration. Thus, the pMHC-NP therapeuticapproach induces clinically significant responses in different diseases(T1D, EAE), animal models and genetic backgrounds (NOD, C57BL/6).

These studies provide evidence of dose-dependent efficacy andfeasibility of treatments for MS using MS-antigen-MHC-nanoparticlecomplexes.

Example 2. Process for Making Antigen-MHC-Nanoparticle Complexes

Inorganic nanoparticles (iron oxide=IONP; gold=GNPs) of a desired size.IONPs are produced via thermal decomposition. IONPs synthesized as suchare biocompatible and can be PEGylated for protein conjugation. To coatpMHC and/or other proteins onto IONPs, surfactant-coated NPs are reactedwith functionalized PEG linkers of the appropriate length. The linkersare purified by HPLC and characterized by ¹H-NMR, MALDI/GPC and GPC, toconfirm chemical identity, purity, molecular weight and polydispersity.Similar linkers and approaches can be used to coat GNPs, except that thelinkers will have a thiol (SH) group at their NP-binding end.

Example 3. Size, Density, and Exposure of pMHC-Coated Nanoparticles

I. Synthesis and Characterization of Gold-Based pMHC-Coated NP.

Gold nanoparticles (GNPs) of specific sizes were synthesized. The size,density, surface charge and monodispersity of the GNP preparations aremeasured using spectrophotometry, transmission electron microscopy (TEM)and dynamic light scattering. The GNP samples are then concentrated andconjugated with mono-specific pMHC complexes using different approachesas described below. Applicants have developed methods to quantitate thepMHC valency per GNP and to concentrate the pMHC-coated GNP preparationsof different sizes at high densities (˜10¹⁴/ml) without compromisingmonodispersion (FIG. 11).

II. Characterization of the pMHC Binding Capacity of GNPs.

pMHC complexes were coated onto GNPs of various sizes using twodifferent approaches: (i) random binding of pMHC to the GNP surface viaelectrostatic interactions; and (ii) directional binding through athiol-PEG-NH₂ linker (in this case, an additional thiol-PEG linker asGNP stabilizer was used to prevent aggregation). It was believed thatthe first approach would enable very high ligand densities (of pMHC perGNP) while compromising the directionality of pMHC binding (i.e. only afraction of the molecules might become available for recognition bycognate T-lymphocytes). The second approach aimed to generatepMHC-coated GNPs carrying lower densities of pMHC but bounddirectionally, via their C-termini. Both approaches were tested on GNPsof various diameters, ranging from 14 to 40 nm. It was confirmed that,for both approaches, the pMHC-binding capacity of GNPs is a function ofsize, and more specifically surface area (higher number of pMHCs onbigger NPs). Surprisingly, it was found that PEG mediated-binding notonly ensures the directionality of binding but also enhances the bindingcapacity of individual GNPs (contrary to initial expectations). Table 1below summarizes the data.

TABLE 1 pMHC binding capacity of GNPs Diameter Surface area: pMHCs/GNPpMHCs/GNP (nm) (×10² nm²) (absorption) (linker) 14 7 212 20 12 3,750 3028 335 40 50 2,850 5,250III. Agonistic activity versus pMHC content.

The effects of pMHC valency, GNP size, GNP density and coating strategyon the functional (agonistic) activity of pMHC-coated GNPs in vitro weretested. The ability of various IGRP₂₀₆₋₂₁₄-K^(d)-GNP preparations toactivate cognate (IGRP₂₀₆₋₂₁₄-specific) naive CD8+ T cells (hereinreferred to as ‘8.3-CD8+ T-cells’) derived from T-cell receptor (TCR)transgenic NOD mice (or 8.3-NOD mice) were compared. The first set ofexperiments aimed to compare the effects of IGRP₂₀₆₋₂₁₄-Kd (pMHC)valency over a range of GNP densities in the culture. GNPs conjugatedwith a control (non-cognate) pMHC complex (Tum-K^(d)) were used asnegative controls. As expected, IGRP₂₀₆₋₂₁₄-K^(d)-coated (but notTUM-K^(d)-coated) GNPs activated these T cells (as measured by IFNγproduction), and they did so in a GNP dose-(hence pMHC dose)-dependentmanner. FIG. 12 shows an experiment using ˜14 nm GNPs coated withdifferent numbers of pMHC molecules/GNP using the linker method. FIG. 12compares the amounts of IFNγ secreted by cognate 8.3-CD8+ T-cells inresponse to two different pMHC-GNP samples (both consisting of ˜2×10¹³GNPs of 14 nm in diameter/ml). Au-022410 and Au-21910 carried ˜250 and˜120 pMHCs/GNP, respectively. Au-011810-C carried ˜120 controlpMHCs/GNP. GNPs coated with ˜2-fold higher numbers of pMHC complexes/GNPhad superior agonistic activity. Thus, the agonistic activity ofpMHC-coated GNPs is a function of total pMHC (GNP) content. Theseresults were counter-intuitive as the state of the art would suggestthat, in the absence of costimulatory molecules on the NPs, increasingthe numbers of pMHCs on individual NPs would also increase avidity andshould promote deletion (cell death), rather than proliferation andcytokine secretion from cognate T-cells. This would be true for both lowavidity and high avidity T-cells. For example, previous work by theApplicants (Han et al., (2005) Nature Medicine 11(6):645-652) and othersindicated that peptides recognized with high avidity or peptidesrecognized with low avidity but given a high concentrations have anincreased ability to delete cognate T cells in vivo. Therefore, in thecontext of therapeutic delivery of intravenous antigen-MHC-coatednanoparticles or soluble peptides, cognate T-cells should undergodeletion in a peptide affinity and dose-dependent manner. Thisexpectation was not met by the data shown in FIG. 12.

IV. A Valency Threshold in the Agonistic Activity ofPeptide-MHC-Nanoparticle Complexes

To further investigate the role of peptide-MHC (pMHC) valency on theagonistic properties of pMHC-conjugated nanoparticles (pMHC-NPs), theability of 8 nm diameter iron-oxide (Fe₃O₄) NPs covalently coupled withincreasing numbers of IGRP₂₀₆₋₂₁₄/K^(d) pMHC monomers, to trigger thesecretion of IFN-gamma (IFNγ) by cognate (IGRP₂₀₆₋₂₁₄/K^(d)-specific)CD8+ T cells (herein referred to as 8.3-CD8+ T-cells) in vitro wascompared. As shown in Table 2, 8.3-CD8+ T cells produced negligibleamounts of IFNγ when cultured in the presence of NPs coated with 8 pMHCmonomers per NP, but produced substantially higher amounts of IFNγ inresponse to NPs coated with higher pMHC valencies, even as low as 11pMHC monomers/NP, in a dose-response manner.

TABLE 2 Secretion of IFNγ by 8.3-CD8+ T cells in response to NPsconjugated with increasing pMHC valencies (at 5 × 10¹¹ NPs/mL)Nanoparticles Core pMHC IFNγ responses (NPs) Core property size (nm)Valency (ng/mL) IGRP-SFPM- Fe3O4 8 8 0.03 110512 IGRP-SFP- Fe3O4 8 110.4 102912 IGRP-SFP- Fe3O4 8 14 0.2 012011 IGRP-SFP- Fe3O4 8 15 0.15031511 IGRP-SFP- Fe3O4 8 31 0.7 051211 IGRP-SFP- Fe3O4 8 39 0.9 100711IGRP-SFP- Fe3O4 8 54 2.3 011411

This positive effect of pMHC valency on the agonistic activity ofpMHC-NPs was maintained over a range of pMHC-NP densities (FIG. 13).Remarkably, however, whereas 25×10¹¹ NPs (per ml) carrying 11 pMHCs/NPhad similar agonistic activity as 5×10¹¹ NPs (per ml) carrying 54pMHCs/NP, increasing the number of NPs carrying 8 pMHCs/NP to values ashigh as 40×10¹¹ NPs/ml had minimal effects (FIG. 14). Taken together,these results indicate that there is a threshold of pMHC valency, lyingbetween 9 and 11 pMHCs/NP, below which relatively large increases in thenumber of NPs (i.e., 5-fold) cannot overcome the low agonistic activityof pMHC-NPs coated at low valencies (it is noted that that the use of>50×10¹¹ NPs in these in vitro experiments is not informative due tocellular toxicity caused by high NP densities).

This pMHC valency threshold effect is further illustrated in FIG. 15,where the IFNγ secretion data are normalized to the concentration oftotal pMHC delivered by the coated NPs in the cultures. NPs carrying 11pMHCs/NP triggered significantly higher IFNγ responses over a range ofpMHC concentrations than those triggered by NPs carrying 8 pMHCs/NP.Furthermore, differences in the agonistic properties of these two NPpreparations increased substantially with total pMHC content. That is,differences in the agonistic properties of 2.4 μg/ml of pMHC deliveredby the NPs as octamers versus monodecamers were much higher thandifferences in the agonistic properties of the same formulations at10-fold lower concentrations of total pMHC.

FIG. 16 shows that these profound effects of pMHC valency on theagonistic properties of pMHC-NPs can also be seen when using larger NPs(which can accept much higher pMHC valencies than the 8 nm NPs studiedin FIGS. 13-15) used at lower NP densities (to normalize the total ironoxide content in the cultures). Whereas 18 nm diameter NPs carrying <10pMHCs/NP had virtually no biological activity up to 4×10¹¹ NPs/ml, theagonistic activity of 18 nm diameter NPs carrying higher pMHC valenciesincreased linearly with NP density. Comparison of FIGS. 15 and 16further shows that 2×10¹¹ 18 nm NPs delivering 61 pMHCs/NP have similaragonistic activity than 2×10¹¹ 8 nm NPs delivering a similar number (54)of pMHCs/NP, indicating that the effects of pMHC valency are notsignificantly affected by NP volume.

Taken together, these data demonstrate that pMHC-coated NPs acquirepowerful agonistic activity above a certain pMHC valency threshold(lying between 9 and 11 pMHCs/NP). Increases in either pMHC valency orNP density can enhance the agonistic properties of pMHC-NPs carrying“threshold” or “supra-threshold” pMHC-valencies but not the agonisticproperties of NPs carrying “infra-threshold” pMHC valencies.

V. Agonistic Activity Versus NP Size and Density.

Further analysis indicated that total pMHC content is not the onlyfactor affecting the agonistic activity of pMHC-NPs in vitro and that NPsize also plays an important independent role. This was investigated bycomparing the agonistic activity of two pMHC-GNP samples of differentsize (14 and 40 nm in diameter, respectively) and different pMHCvalencies but under conditions of similar total pMHC content. In theexperiment shown in FIG. 17, 14 nm GNPs carrying ˜200 pMHCmolecules/GNP, and 40 nm GNPs carrying ˜5,000 pMHCs/GNP were used. TheGNP densities of these two samples was adjusted (to 3×10¹³ and 10¹²GNPs/mL, respectively) to adjust the total pMHC content in each sampleto ˜450 ug/ml. Notably, 8.3-CD8+ T cells responded significantly betterto the 14 nm pMHC/GNP compound than to the 40 nm one over a range oftotal pMHC contents, despite the fact that the latter were decoratedwith significantly more pMHC complexes than the former. This suggestedthat GNP density (more GNPs/cognate T-cell) is key. In other words, 4×40nm NPs carrying 1000 pMHCs/GNP (4000 pMHCs) would be less desirable than40×10 nm NPs carrying 100 pMHCs/GNP (4000 pMHCs). Thus, when takentogether these data suggest that optimal pMHC-GNP preparations are thosecomprised of small GNPs used at high pMHC densities. Increasing pMHCvalency on these small NPs further increase their surprising andunexpected agonistic properties.

VI. Agonistic Activity Versus pMHC Exposure.

As noted above, the pMHC-coated GNP samples are produced by co-coatingGNPs with a 3.4 kD thiol-PEG-NH₂ linker (as acceptor of pMHCcarboxitermini) with a thiol-PEG linker that functions as GNPstabilizer. To investigate if the length of the stabilizing thiol-PEGlinker influences its GNP anti-aggregation properties, the ability ofthe thiol-PEG-NH₂ linker to bind pMHC molecules and/or the agonisticproperties of pMHC-coated GNPs, pMHC-coated GNPs prepared usingstabilizing linkers of different sizes (2 kD and 5 kD, shorter andlonger than the pMHC-acceptor linker, respectively) were compared. Itwas found that both linkers had similar anti-aggregation properties, andthat the 5 kD linker did not inhibit binding of pMHC to the shorter 3.4kD thiol-PEG-NH₂ linker. Notably, however, pMHC-GNPs that were protectedby the shorter (2 kD) thiol-PEG had superior agonistic activity in vitrothan those co-coated with the longer (5 kD) thiol-PEG (FIG. 18). Thissuggests that long protective thiol-PEG linkers shield pMHC moleculesbound to the acceptor linker from exposure to cognate T cells.

VII. Small NPs Covalently Coupled to High Densities of pMHC AffordMaximum Autoregulatory T-Cell Expansion Effects In Vivo.

Nanoparticles having an average diameter of about 10 nm and coupled toeither NRP-V7/K^(d) (also referred to as IGRP₂₀₆₋₂₁₄-K^(d)) or TUM/K^(d)(control) were made in accordance with the methods described herein, andtested for their ability to induce expansion of cognate autoregulatoryCD8+ T cells in vivo. FIG. 19 shows the results of an experiment inwhich antigen-MHC-GNPs were injected intravenously into 10 week-oldwild-type NOD mice bi-weekly for 5 consecutive weeks. Changes in thesize of the cognate T-cell population in the circulation and differentlymphoid tissues in response to therapy were assessed by staining cellsuspensions with fluorescently-labeled antigen-MHC tetramers (bothcognate as well as irrelevant control tetramers). Administration of10-100 fewer GNPs than what was has previously been shown in the art(See, for example, Tsai et al. (2010) Immunity 32(4):568-580) in whichnanoparticles coated with 1-8 pMHCs were tested) but coated with 150antigen-MHCs per GNP resulted in substantially higher expansions (FIG.19). They expanded CD8+ T-cells in vivo to levels several fold higher(up to 44% of all circulating CD8+ T-cells) than those Applicantstypically obtain with nanoparticles coated with a pMHC at a valency ofabout 8 (1-2% cells in blood; See, for example, Tsai et al., Immunity,2010, FIG. 1C). The above data indicate that small nanoparticles coatedwith high antigen-MHC valencies afford maximum T-cell expansion effects.These results were unexpected. Accordingly, it is not the overallavidity of the pMHC-NP-T-cell interaction that is responsible fortherapeutic effect, but rather the avidity of the precursor populationthat gives rise to the T-cells that expand in response to pMHC-NPtherapy. This interpretation is consistent with the data describedherein and implies that valency of pMHCs on NPs should increase thetherapeutic efficacy of pMHC-NPs.

Example 4. Large Expansion of Cognate CD8+ T-Cells by pMHC-GNPs Coatedat Higher pMHC Valencies

It was next determined whether pMHC-NPs have the potential to inducemassive expansions of cognate T-cells in vivo. This was done by treatingmice with several injections of 3×10¹² 10-14 nm NPs carrying 25 ug oftotal pMHC (˜150 IGRP₂₀₆₋₂₁₄/Kd molecules per NP). As shown in FIG. 20,mice treated with 10 doses (twice a week for 10 week) displayed massiveexpansions of cognate IGRP₂₀₆₋₂₁₄ (NRP-V7)-reactive CD8+ T-cells inperipheral blood as compared to their untreated counterparts (from <0.4to >17 or 47% CD8+ T-cells) (lower panels). Such expansion was alreadyseen in a mouse that was sacrificed after 4 doses of pMHC-NPs (upperpanels). The pMHC-NP-expanded cells specifically bound cognate but notnon-cognate pMHC tetramers (NRP-V7/K^(d) vs. TUM/K^(d), respectively).

Example 5. Preparation of pMHC Conjugated Gold NanoParticles

pMHC Conjugated Gold NanoParticle Preparation (pMHC-GNPs, 12 and 30 nm).

Preparation of GNPs.

GNPs were prepared by heating D.D. water (200 mL) in a ball flask in asilicon oil bath till boiling. A solution of 1% HAuCL₄ (4 mL) was thenadded into boiling water. The solution was stirred for 10 min beforeadding of 1% Na Citrate solution. For 12 nm GNPs, 12 mL Na Citratesolution was added. For 30 nm GNPs, 12 mL Na Citrate solution was added.A wine color appears immediately after adding Na Citrate solution. Tocomplete the reaction, GNP solution was stirred for 30 minutes more.This is a modification of the method described in Levy, R. et al.(“Rational and combinatorial design of peptide capping ligands for goldnanoparticles.” J Am Chem Soc 126, 10076-84 (2004)) which is hereinincorporated by reference.

Surface Modification of GNPs.

GNPs were pegylated by addition of 25 mM thiol-PEG-NH₂ (M.W. 3,400) and50 mM thiol-PEG (M. W. 2,000, PEG/GNP ratio 10,000:1) into GNP solution.The solution was stirred for 5 hours at room temperature. Pegylated GNPswere then washed with 3×30 mL sterilized D. D. water to remove excessPEGs, and resuspended in 40 mL of 100 mM MES (C₆Hi₃NO₄S.xH₂O) buffer, pH5.5.

pMHC Conjugation.

pMHCs (IGRP₂₀₆₋₂₁₄/Kd, 4 mg) was added into solution of pegylated GNPs,drop-by-drop with mild stirring at room temperature. The mixture isstirred for one hour before the addition of 20 mg1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The mixture isstirred for additional 4 hrs. pMHC-GNPs conjugates are then washed with40 mL Phosphate Buffered Saline (PBS, PH 7.2-7.4) for three times, andresuspended in 8 mL PBS.

Example 6. Preparation of pMHC Conjugated Gold NanoParticles

Preparation of pMHC Conjugated GNPs (pMHC-GNPs, 2-10 nm).

Prepare GNPs (2-5 nm).

GNPs of 2-5 nm were prepared by dissolving 250 mg (for 2 nm GNPs) or 50mg (for 4 nm GNPs) Dodecylamine in 10 mL of DDAB solution (100 mMDidodecyldimethylammonium bromide (DDAB) in Toluene). Secondly, 100 mgTetrabutylammonium borohydride (TBAB) was dissolved in 4 mL of DDABsolution. Solutions of Dodecylamine and TBAB were then mixed in a 50 mLthree-neck flask, stirring under nitrogen. 34 mg AuCl₃ was resolved in4.5 mL DDAB solution, and injected quickly into a mixture of TBAB andDodecylamine solution. Solution becomes deep red immediately, indicatingthe formation of GNPs. The mixture was continuously stirred for 30 min,and 15 mLs of ethanol were added into the mixture. The mixture was thenspun at 4,100×g for 12 min to precipitate GNPs.

Prepare GNPs (6-10 nm).

To prepare GNPs of 6-10 nm Decanoic acid (172 mg) was first dissolved in10 mL Toluene, and then mixed with various amounts of TBAB solution (4and 1 mL for 6 and 10 nm GNPs, respectively) in a 50 mL three-neckflask, when stirring under nitrogen. AuCl₃ (34 mg dissolved in 4.5 mLDDAB stock solution) was then quickly injected into the mixture of TBABand Decanoic acid solution. The solution became deep red immediately.The mixture was continuously stirred for 30 min, and 15 mL ethanol wasadded into the mixture. The mixture is then spun at 4,100×g for 12 minto precipitate GNPs.

Surface Modification of GNPs.

GNPs were resuspended in 20 mL of 0.1 M mercaptopropanoic acid (MPA) inmethanol, pH 10 and stirred for one hour at room temperature. 10 ml,ethyl acetate was then added. The mixture was then spun at 4,100×g for15 min. The precipitated GNPs were then washed with 30 mL sterilizedD.D. water for three times, and resuspended in 20 mL 100 mM MES(C₆H₁₃NO₄S.xH₂O) buffer, pH 5.5. To this mixture, solutions of 0.5 MPolyoxyethylene bis(amine) (at 10,000:1 PEG/GNP ratio) and 0.1M1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (final EDCconcentration 2 mM) were added. The mixture was then stirred for 4hours. The pegylated GNPs were washed with 3×30 mL sterilized D.D. waterto remove excess PEG and EDC.

pMHC Conjugation.

Pegylated GNPs were resuspended in 20 mL 100 mM MES (C₆Hi₃NO₄S.xH₂O)buffer, pH 5.5. pMHCs (5 mg/mL, total 10-30 mg) were then added toresuspended GNPs (500:1 pMHC/GNP ratio), drop-by-drop, and stirred for 1hour at room temperature before adding 0.1M1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (final EDCconcentration 2 mM). The mixture was stirred for 4 more hours. pMHC-GNPsconjugates were washed three with 40 mL Phosphate Buffered Saline (PBS,PH 7.2-7.4), and then resuspended in 10-20 mL PBS.

pMHC Optimization.

The optimal pMHC-NP design consists of small particles coated with pMHCmonomers at the highest possible density, which yields pMHC complexesseparated by 3-4 nm. pMHC-NPs designed following these principles haveoptimal potency (maximal agonistic activity and Treg expandingproperties at lower doses of total pMHC). This has been confirmedexperimentally for the findings for class I pMHC-Nps is also true forpMHC class II-coated Np. When used at optimal density and dose of pMHC,both pMHC class I and class II-coated NPs, can expand cognateautoregulatory T-cells. Thus, the ideal pMHC-NP design involves theability to deliver densely packed pMHCs on NPs.

This is supported by two experimental observations: (1) larger NPscoated with similar number of pMHCs than their smaller counterparts,particularly at threshold valency values (i.e. 10 pMHCs/NP) weresignificantly less agonistic, independently of total pMHC input, thethreshold value spacing individual pMHCs at 10 nm requires 60 pMHCs/NPand >120 pMHCs to reach the 3-4 nm spacing distance; and (2) small NPscoated at very high densities of pMHC has the highest agonisticactivity, also independently of total pMHC input. Thus, reductions inthe pMHC distance (i.e. from the 10 nm threshold distance to 2-4 nm) byincreasing pMHC density increases the overall avidity and TCR signalingcapacity of the pMHC-NP-T-cell interaction. Thus, the threshold foragonistic activity is defined by pMHC molecular density (pMHCintermolecular distance) on the NP surface rather than by pMHC molecularnumber.

Collectively, this lays the groundwork for the optimal design of pMHC-NPformulations aimed at expanding autoantigen-specific regulatory T-cellsin vivo for the treatment of autoimmune disorders. Applicant has shownthat optimal formulations can yield massive expansions ofautoantigen-specific regulatory T-cells in vivo to frequencies as highas 1 in 2 circulating CD8+ or CD4+ T-cells at extremely low doses ofpMHC-NPs. Since therapeutic levels are significantly lower than thoseinducing these massive expansions, this approach benefits from a largesafety and efficacy margin that should enable the treatment ofaggressive autoimmune conditions.

It should be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this invention. The materials, methods, andexamples provided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Allpublications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Sequence Listing

SEQ ID NO: 1: pMOG₃₈₋₄₉ antigen: GWYRSPFSRVVH.SEQ ID NO: 2: Protein sequence of vector comprising pMOG₃₈₋₄₉ antigen(FIGS. 9A-9B).SEQ ID NO: 3: DNA sequence of vector comprising pMOG₃₈₋₄₉ antigen (FIGS.9A-9B).

1.-28. (canceled)
 29. A method for treating multiple sclerosis or amultiple sclerosis related disorder in a subject in need thereofcomprising administering to the subject an effective amount of anantigen-MHC-nanoparticle complex, wherein the antigen-MHC-nanoparticlecomplex comprises: a nanoparticle core and multiple sclerosis-relatedantigen-MHC protein (pMHC) complexes operatively coupled to thenanoparticle core, wherein the nanoparticle core has a diameter fromabout 1 nm to about 100 nm and wherein the pMHC density on thenanoparticle core comprises from about 0.005 pMHC/100 nm² to about 25pMHC/100 nm².
 30. (canceled)
 31. (canceled)
 32. The method of claim 29,wherein the nanoparticle core has a biodegradable layer on the outersurface of the nanoparticle core and the pMHC complexes are operativelycoupled to the nanoparticle core or the biodegradable layer on thenanoparticle core.
 33. The method of claim 29, wherein the multiplesclerosis-related antigen of the pMHC complexes is an antigen derivedfrom a protein selected from the group of myelin basic protein, myelinassociated glycoprotein, myelin oligodendrocyte protein, proteolipidprotein, oligodendrocyte myelin oligoprotein, myelin associatedoligodendrocyte basic protein, oligodendrocyte specific protein, heatshock proteins, oligodendrocyte specific proteins NOGO A, glycoproteinPo, peripheral myelin protein 22, and 2′3′-cyclic nucleotide3′-phosphodiesterase and myelin oligodendrocyte glycoprotein (MOG) or anantigen corresponding to a peptide having at least 80% identity to apeptide comprising the sequence of any one of SEQ ID NOS: 1 and 4-17.34. The method of claim 29, wherein the nanoparticle core isnon-liposomal.
 35. The method of claim 29, wherein the nanoparticle corecomprises a metal, a metal oxide, a metal sulfide, a metal selenide, amagnetic material, a polymer, iron, iron oxide, or gold.
 36. The methodof claim 32, wherein the biodegradable layer comprises one or more ofdextran, mannitol, or poly(ethylene glycol).
 37. The method of claim 29,wherein the pMHC complexes are covalently linked to the nanoparticlecore.
 38. The method of claim 32, wherein the pMHC complexes arecovalently linked or non-covalently linked to the nanoparticle core orthe biodegradable layer.
 39. The method of claim 29, wherein the pMHCcomplexes are covalently linked to the nanoparticle core through alinker less than 5 kD in size.
 40. The method of claim 32, wherein thepMHC complexes are covalently linked to the nanoparticle core or thebiodegradable layer through a linker less than 5 kD in size.
 41. Themethod of claim 39, wherein the linker comprises polyethylene glycol.42. The method of claim 29, wherein the nanoparticle core isbioabsorbable and/or biodegradable.
 43. The method of claim 29, whereinthe MHC of the pMHC complexes is a MHC class I protein.
 44. The methodof claim 43, wherein the MHC class I protein comprises all or part of aHLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G or CD-1 protein
 45. The methodof claim 29, wherein the MHC of the pMHC complexes is a MHC class IIprotein.
 46. The method of claim 45, wherein the MHC class II protein ofthe pMHC complexes comprises all or part of a HLA-DR, HLA-DQ, or HLA-DPprotein.
 47. The method of claim 29, wherein the ratio of the number ofpMHC complexes to the nanoparticle core is from about 10:1 to about500:1.
 48. The method of claim 29, wherein the multiplesclerosis-related disorder is selected from the group consisting ofneuromyelitis optica (NMO) and neuropathic pain.
 49. The method of claim29, wherein the nanoparticle core has a diameter from about 1 nm toabout 50 nm.
 50. The method of claim 29, wherein the nanoparticle corehas a diameter from about 1 nm to about 20 nm.
 51. The method of claim29, wherein the nanoparticle core has a diameter from about 5 nm toabout 20 nm.
 52. The method of claim 29, wherein the nanoparticle corehas a diameter from greater than 15 nm to about 100 nm.
 53. The methodof claim 29, wherein the nanoparticle core has a diameter from greaterthan 15 nm to about 50 nm.
 54. The method of claim 29, wherein thesubject is a mammal.
 55. The method of claim 33, wherein the multiplesclerosis-related antigen comprises a polypeptide sequence identical tothat set forth in any one of SEQ ID NOs: 1 and 4-17.
 56. The method ofclaim 29, wherein the method develops or expands anti-inflammatory CD4+T cells.
 57. The method of claim 32, wherein: the nanoparticle corecomprises a metal, a metal oxide, a metal sulfide, a metal selenide, amagnetic material, a polymer, iron, iron oxide, or gold; thebiodegradable layer comprises one or more of dextran, mannitol, orpoly(ethylene glycol); the pMHC complexes are covalently linked to thebiodegradable layer; the MHC is a MHC class II protein selected from oneor more of all or part of a HLA-DR, HLA-DQ, or HLA-DP protein; whereinthe ratio of the number of pMHC complexes to the nanoparticle core isfrom about 10:1 to about 500:1; the multiple sclerosis-related disorderis selected from the group consisting of neuromyelitis optica (NMO) andneuropathic pain; and the nanoparticle core has a diameter from about 1nm to about 50 nm.